The NCMI is dedicated to the advancement of electron cryo-microscopy (cryo-EM) methodology for structure determination of nanomachines in their various functional states at the highest possible resolutions. Having established four state of the art electron cryomicroscopes and CCD cameras and accomplished de novo backbone traces of proteins in both viruses and molecular chaperonins, our Center is well poised to continue our research mission, including 4 core projects, 12 collaborative projects, 14 service projects, workshops, dissemination, training and maintaining an advisory board. In the core projects, we will 1) extend the resolution of single particle reconstruction beyond 4 A;2) explore Zernike phase contrast for biological cryo-EM at subnanometer resolution;3) develop techniques for "single particle" averaging in electron cryotomography at maximum possible resolution;4) extend our data management infrastructure to fully document large scale cryo-EM datasets and processing. Specific aim 1 will bring a new level of capabilities to cryo-EM, providing molecular structures with a level of detail comparable to x-ray crystallography. The second aim will demonstrate the potentials and limitations of imaging with a theoretically promising new type of electron optics. Our third aim is targeted at uncovering unprecedented structure details about dynamic nanomachines operating in a native or near-native environment. The fourth aim will allow us to mine cryo-EM data easily to improve current methodologies and data sharing. The collaborative and service projects will cover a broad spectrum of specimens including viruses, molecular chaperonins, RNA, membrane and membrane associated proteins, and macromolecular assemblies associated with or inside cells. We will undertake a new effort in identifying projects of biomedical and translational relevance, through interactions with clinical and translational investigators. We will disseminate our software, experimental and computational protocols via workshops and web based materials. We will continue our tradition of training both local and distant students, postdocs and new investigators. We will maintain a dynamic membership of our advisory board. These efforts will extend cryo-EM's capabilities both at very high resolution at the interface with x-ray crystallography and at lower resolutions, relating macromolecular structures in the cell or cell-like environment. PUBLIC HEALTH RELEVANCE: Our proposed cryo-EM methodology is targeted to study structures of biologically active nanomachines which are potential drug targets for treating or preventing diseases. Our collaborative research covers nanomachines closely tied to infectious diseases (viruses, bacteria and parasite), neurodegenerative diseases and aging (molecular chaperonins), cancer (complex involving cellular processes and signaling), and cardiovascular diseases (lipoprotein, blood clotting factors and ion channels).